EFFECT of the KANAMYCIN RESISTANCE MARKER on STABILITY of 2M-BASED EXPRESSION PLASMIDS
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Arch. Biol. Sci., Belgrade, 59 (1), 1-12, 2007. DOI:10.2298/ABS0701001S EFFECT OF THE KANAMYCIN RESISTANCE MARKER ON STABILITY OF 2m-BASED EXPRESSION PLASMIDS STANKOVIĆ NADA., VASILJEVIĆ BRANKA., and†G. LJUBIJANKIĆ Institute of Molecular Genetics and Genetic Engineering, 11010 Belgrade, Serbia Abstract – In this paper we describe the effect of the kanamycin resistance gene (Kmr) on 2μm-based plasmid mainte- nance in Saccharomyces cerevisiae. The influence of this marker gene on the loss of the stable model-vectors proved to be constant, as well as independent of carbon source and culture growth rates. In strains for GALUAS – driven heterolo- gous protein production introduction of Kmr resulted in curing of the yeast episomal plasmid (YEp) from the population in a small number of generations. Application of selective pressure on the strain producing recombinant penicillin G ami- dase (rPGA) did not provide the expected increase of protein yield. The influence of genetic elements for heterologous protein production on vector stability was examined, and the most destabilizing factors prove to be the presence and expression of the foreign gene. Key words: 2μm plasmid, kanamycin resistance, penicillin G amidase, plasmid stability, Saccharomyces cerevisiae, selective markers. UDC577.182.76 : 577.21 579.25 INTRODUCTION based expression vectors were successfully applied in our laboratory for expression of the recombinant penicillin G Expression vectors based on the naturally occurring 2μm amidase (penicillin acylase, PGA, pac) gene from the plasmid are the sole vectors in baker’s yeast which satis- bacterium Providencia rettgeri (Ljubijankićet al., fy two major biotechnological requirements: mitotic sta- 1999; Ljubijankićet al., 2002) and gene for human bility and high copy number. Vectors that employ the interferon b (Todorović et al., 2000) in baker’s whole 2μm sequence have proved to be the most stable yeast. YEps (yeast episomal plasmid), since they contain all the sequences that control plasmid DNA replication and par- During our work with various Yeps, we realized the titioning. However, artificially constructed 2μm-based necessity for quick and exact monitoring of the mitotic vectors appear to be less stable than native 2μm plasmids, stability of plasmids. For vectors whose presence is diffi- and are maintained at a lower copy number (Futcher cult or expensive to detect via a recombinant product, the and Cox, 1984). Alternative methods in yeast expres- introduction of a dominant selective marker is a logical sion vector design include disintegrative vectors, which alternative. Despite much effort to introduce novel mark- are recombination cointegrants made up of a 2μm-based er genes (as listed in van den Berg and vector and pRL (Chinerry and Hinchliffe, Steensma, 1997) into yeast biotechnology, there are 1989). In the yeast cell, the cointegrant is resolved by few real dominant selective markers in S. cerevisiae, and (recombinase)-mediated FRT recombination into its two their application is restricted. Although the majority of components. An Escherichia coli plasmid carrying the selective markers cannot be applied for large-scale pro- yeast LEU2 marker and a single FRT (FLP recognition duction, their role in construction and selection of the target) sequence (Bruschi and Howe, 1988), pRL is optimal productive clones is irreplaceable. In order to subsequently lost from the population because it is retain a high yield of biomass, we decided to introduce unable to propagate in S. cerevisiae. Disintegrative 2μm- the dominant selective marker gene for aminoglycoside 1 2 N. STANKOVIĆ ETAL. phosphotransferase I (APT I, Kmr), which confers resist- BLITZ cassette is driven by the strong and strictly regu- ance to kanamycin in prokaryotes and G418 in eukary- lated GAL1-10 promoter, which is repressed in the pres- otes (Jimenez and Davies, 1980). The ence of glucose and induced about 1000-fold by galac- KanMX4 cassette (Wach et al., 1996) that we used has tose (Romanos et al., 1992). a dual (bacterial/fungal) promoter. The strong constitu- tive TEF promoter from the fungus Ashbya gossypii The aim of this study was to test the dominant mark- er gene for G418 resistance as a useful tool for monitor- allows effective expression of the Kmr gene in yeast. A ing the proportion of plasmid-bearing cells in culture, single copy of Kmr in A. gossypii confers resistance for while simultaneously examining the influence of the up to 8 mg/ml of geneticin (Steiner and selective marker on YEp stability. Philippsen, 1994). However introducing a new gene could burden plas- MATERIALS AND METHODS mid expression and plasmid mitotic stability, consequent- ly decreasing the yield of the recombinant product. In Bacterial and yeast strains order to examine the effect(s) of the dominant selective – marker Kmr on vector stability, various E.coli/S. cerevisi- The E. coli strain DH5a (F Dlac U169 (F80 lacZ ae shuttle vectors were constructed based on disintegra- DM15) supE44 hsdR17 recA1 gyrA96 endA1 thi-1 relA1) tive plasmids pBLU-D (Ludwig and Bruschi, (Hanahan 1983) was used to clone all the plasmids and 1991) and pGoB-2 (Ljubijankić et al., 1999). constructs described in this work. The S. cerevisiae strain Plasmid pBLUR-D, with the yeast auxotrophic URA3 CBL1-30 (MATα [cirº] pep4-3 his3Δ::GAL10p-GAL4- marker gene, is the resolved form of pBLU-D (Fig. 1) URA3 leu2-3,112 trp1-289 ura3-52 canR), harboring and appears to be maintained at a high copy number with- plasmids pGoBR-2KS and pGIFNR (Todorović et al., out selective pressure. Construct pGoB-2 (Fig. 2) is the 2000), has been described previously (Ludwig 1991). construct for pac expression in which maximal recombi- Isogenic ura– strain GSP-3 (MATα [cirº] pep4-3 nant enzyme yield is provided by the BLITZ expression his3Δ::GAL10p-GAL4-ura3Δ247 leu2-3,112 trp1-289 cassette (Ludwig et al., 1993). Expression from the ura3-52 canR) was constructed as described previously r Fig. 1 - pBLU-based disintegrative vectors. The Km gene was cloned in the SmaI site of pBLU-D with or without different TRPter sequences in dif- ferent orientations. The ure of pBLU-D was provided by courtesy of C. V. Bruschi. EFFECT OF KANAMYCINE RESISTANCE MARKER 3 Fig. 2 - 2mm based vectors with the BLITZ expression cassette: a- Vector pBLAST gained after removing the lacZ reporter gene from pBLITZ con- tains the “empty” BLITZ expression cassette with SmaI and BamHI sites for foreign gene insertion (Ludwig et al., 1993). b - Different sequences inserted into the expression cassette resulted in different expression vectors. The 3’f pa - pac gene was cloned together with the downstream fragment of the P. rettgeri chromosome. The figure of pBLAST was provided by courtesy of C. V. Bruschi. (Pavković & Ljubijankić, 2000) in order to Media and culture conditions provide an appropriate genetic background for testing the plasmid stability of URA3 carrying pBLU-based vectors. Esherichia coli strains were grown in LB medium Briefly, the URA3 gene on the CBL1-30 chromosome with or without agar (20 g/L). Ampicillin was added at was replaced with the inactive allele ura3D247, which 100 µg/µl and kanamycin at 60 µg/µl for selection of was obtained by deletion of the 247 bp StuI/EcoRV frag- recombinant plasmids. Saccharomyces cerevisiae strains ment of the URA3 gene on the YIplac211 plasmid were grown in one of the following standard yeast media: (Gietz and Sugino, 1988). The inactive allele semidefined rich YPD (1% yeast extract, 2% peptone, was introduced into competent CBL1-30 yeast cells by 2% glucose), YPRaf (1% yeast extract, 2% peptone, 2% electroporation. The Ura– transformants were scored on raffinose), and YPGal (1% yeast extract, 2% peptone, 2% galactose), with or without addition of 200 µg/µl of G418 DOura– medium with 5-fluoroorotic acid (Boeke et (geneticin, G418 sulfate Gibco BRL, Gaithersburg, MD); al., 1984). 4 N. STANKOVIĆ ETAL. and the defined yeast synthetic complete media, leucine- described by Inoueet al. (1990). Yeast transformation less (DOleu–) and uracil-less (DOura–), described by by electroporation was performed according to Sherman et al. (1986). For pac expression, a self- Meilhocet al. (1990). The transformation efficiency inductive medium was used (YPGal with addition of of S. cerevisiae was in the order of magnitude of 103 0.2% glucose). transformants/mg of DNA. Escherichi coli plasmid DNA was isolated using the QIAGEN Plasmid Kit and the DNA transformation and recombinant DNA techniques QIAGEN Mini-prep Kit (QIAGEN) or as described by Del Sal et al. (1989). Manipulations of DNA such as The DH5α E. coli strain was transformed as restriction enzyme digestions, ligations and agarose gel Table 1. List of plasmids used in this work. a See text for details. E. coli vectors Plasmid Relevant characteristica Source or reference: pFAKanMX4 Apr, Kmr Wach et al., 1996 pUC18/19 Apr Yanisch-Perron et al., 1985 r pUC19T Ap , S. c. TRP1ter Storici, F. r r pUC18TK Ap , S. c. TRP1ter, Km this paper r r pUC19TK Ap , S. c. TRP1ter, Km this paper r r pUC18KT5 Ap , S. c. TRP5ter, Km this paper r r pUC19KT5 Ap , S. c. TRP5ter, Km this paper E. coli / S. cerevisiae shuttle vectors Plasmid Relevant characteristica Source or reference: pBLU-D pRL + URA3 Ludwig and Bruschi, 1991 pBLU-KP pRL + URA3, Kmr this paper pBLU-KS pRL + URA3, Kmr this paper r pBLU-KT5P pRL + URA3, Km , TRP5ter this paper r pBLU-KT5S pRL + URA3, Km , TRP5ter this paper r pBLU-TK pRL + URA3, Km , TRP1ter this paper r r YCpKan pRL + Ap , Km , BLITZ cassette, TRP5ter Todorović et al., 2000 pGIFN pRL + Kmr, BLITZ cassette, hIFN-b Todorović et al., 2000 pGoB-2 pRL + BLITZ cassette, P. r. pac Ljubijankićet al., 1999 pGoB-2KS pRL + Kmr, BLITZ cassette, P. r. pac Todorović et al., 2000 EFFECT OF KANAMYCINE RESISTANCE MARKER 5 electrophoresis were performed as described by r 2681 bp BglII/SalI Km TRP5ter cassette from YCpKan Maniatis et al.